The female reproductive tract is responsible for producing endocrine hormones, developing mature, healthy gametes (oocytes) and providing the site for fertilization and an environment that supports fetal development. There are five main organs in the female reproductive tract-the ovary, fallopian tubes, uterus, cervix and vagina. Each organ is responsible for unique aspects of reproductive function, but act integrally to support overall endocrine health, fertility, and fetal development. The reproductive tract organs are assembled from multiple cell lineages to create individual follicles (that enclose and support oocytes), oviductal/fallopian tubes, uterine myometrium and endometrium, the cervix and the vagina. Traditionally, research of the female reproductive tract has relied on two-dimensional (2D) cultures of isolated primary cells or immortalized cell lines grown on plastic and independent of adjacent cells, tissue architecture, and functional context. Moving to a three-dimensional (3D) culture environment has allowed us to better understand the function and interaction of cells within individual organs and interrogate interactions between tract tissues in co-cultures (e.g., the follicle and the ovarian surface cells, or the uterine myometrium and endometrium) to measure responses to normal reproductive hormones, pathologic conditions (such as high levels of androgens) or exposure to endocrine disruptors. New biomaterials and 3D culture systems have now presented us with the exciting opportunity to create a complete in vitro reproductive tract whereby each of the cultured organs can be assembled into a linked perfusion culture system. Just as the biological function and responses of 2D monolayer cell cultures differ from those of 3D-cultured organoids, we predict that the biology of the reproductive organs when studied in an integrated series will more closely recapitulate the in vivo environment. In Aims 1 and 2, we propose to develop in vitro cultures of human reproductive tissues that phenocopy in vivo function in terms of hormone production and response to the physiologically relevant reproductive hormones follicle-stimulating hormone (FSH) and estrogen. We will use the 3DKUBE culture platform (KIYATEC), which not only permits control of perfusion to mimic tissue circulation, automated sampling for pharmacokinetic analyses, tissue imaging and in situ bioassays, but also will facilitate integration of the individal organ cultures into a functional in vitro female reproductive tract culture system in Aim 3. The successful development of an ex vivo female reproductive tract will give us the unique ability to interrogate normal hormonal responses of each organ in the context of the complete reproductive tract, as well as examine responses of the organs and system to agents that pose reproductive hazards. Toxicologic testing on female reproductive function and fertility is currently limited to animal studies. Our proposed Ex Vivo Female Reproductive Tract Integration In a 3D Microphysiologic System would permit earlier assessment of the effects of drugs, toxicants or vaccines on the human female reproductive system prior to exposure in clinical trials. PUBLIC HEALTH RELEVANCE: The female reproductive tract is an integrated set of organs that supports women's overall endocrine health, fertility and fetal development. Each organ within the tract is composed of different cells that interact with each other, which relies on a precise tissue architecture that can be more effectively studied in three-dimensional (3D) tissue cultures. Our goal is to develop and validate 3D culture systems of the five major organs of the reproductive tract into an Ex Vivo Female Reproductive Tract Integration In a 3D Microphysiologic System that can be used to measure responses to normal hormones, endocrine disruptors, and other reproductive hazards and in the drug development pipeline.